Genomics|Activities|FBR|HH|Clinical Validity|Page 1

Summary

Among non-Hispanic Caucasians, clinical sensitivity of C282Y homozygosity for detecting individuals with primary iron overload and associated morbidity is estimated to be at least 87 percent (95 percent CI 80 to 94%).
- It is based on four studies totaling 247 individuals, not all of whom were symptomatic
- When the disorder is defined more rigorously in clinical terms, clinical sensitivity of the test increases
Actual clinical sensitivity is likely to be slightly lower, because analytic sensitivity is less than 100 percent (estimated in Question 9 to be 98.4 percent).
It is not possible to confidently estimate clinical sensitivity among other racial/ethnic groups because little, if any, data are published
Among other racial/ethnic groups, the sensitivity appears to be lower
- 0% among Hispanic Caucasians according to 6 cases reported in 1 studies
- 0% among Black/African Americans according to 14 cases reported in 1 studies
- 0% among Asians/Asian Americans according to 23 cases reported in 2 studies

Introduction
The definitions of clinical sensitivity (Question 18) and clinical specificity (Question 19) can be derived using a two-by-two contingency table for data from case/control or cohort studies. If the data are from a general population cohort, both positive predictive and negative predictive values (Question 23) can also be directly computed. In Table 3-1, the rows are defined by the HFE gene test results, stratified into two categories; C282Y homozygosity and all other test results. The HFE gene has been identified as the major genetic factor leading to iron overload in the Caucasian population, and the C282Y mutation is, by far, the most important mutation in this gene. The columns are defined by the specific clinical disorder that the screening test aims to detect – in this instance, primary iron overload with associated morbidity. The first column contains all individuals with primary iron overload sufficient to cause significant morbidity and mortality, and the second column contains all individuals who do not have the clinical manifestations of iron overload.

Definition of clinical phenotype
In Question 1, a general definition of the disorder being screened for is stated as: primary iron overload of adult onset sufficient to cause significant morbidity and mortality. This definition includes individuals who will develop clinical manifestations of iron overload in their adult life. A more specific definition of the clinical phenotype for primary iron overload is needed before selecting studies that provide data appropriate for assessing clinical validity.

Clinical sensitivity of C282Y testing for primary iron overload
Clinical sensitivity refers to the proportion of individuals who have, or who are destined to develop, the primary iron overload phenotype who have a positive test result for C282Y homozygosity. In contrast, analytic sensitivity describes how often the laboratory correctly identifies C282Y homozygosity. The penetrance of this genotype – or the proportion of individuals homozygous for C282Y who will progress to the primary iron overload phenotype - is not yet precisely known, but is recognized to be considerably below 100 percent.

The ideal study to assess clinical performance
The ideal study to assess clinical performance of HFE testing as a way to detect the primary iron overload phenotype would be to perform DNA testing in a large population-based cohort of young adults. This entire population would then be followed at intervals to determine in whom, and at what age, the phenotype of interest developed. At the conclusion of the study, it would be possible to fill in the four critical numbers in Table 3-1. Such a study would not provide the information in a timely manner and also would not be considered ethically acceptable.

A realistic study to assess clinical sensitivity
A more realistic approach would be to first identify a group of individuals who have the primary iron overload phenotype (A+C from Table 3-1), and then determine the proportion who are C282Y homozygotes (A from Table 3-1). This would provide an estimate of clinical sensitivity, but this design does not allow for the computation of the positive predictive value (the penetrance) of the genotype. Case or case-control studies can be used to determine the proportion of individuals clinically affected with the primary iron overload phenotype who are C282Y homozygotes. Limitations of this approach include:

Clinical sensitivity in non-Hispanic Caucasians in the United States
Initially, the focus of this analysis is the non-Hispanic Caucasian population, because most of the iron overload in this group is associated with the HFE gene. This review uses the term ‘non-Hispanic Caucasian’ as a surrogate for the more common designation of ‘northern European Caucasian’. Few, if any, studies published in the U.S. collect information about country of origin, but many collect information about race/ethnicity. A total of 10 studies report the frequency of the C282Y homozygous genotype in non-Hispanic Caucasian individuals previously classified as having the primary iron overload phenotype, based on biochemical and/or clinical evidence. Definitions of the primary iron overload phenotype are variable. Appendix A contains a summary table of all 10 studies. Overall, the clinical sensitivity ranges from 32 to 91 percent (consensus 69%) and is highly heterogeneous (c2=155, p< 0.001). Appendix A has complete information on these estimates.

In order to properly examine the relationship between C282Y homozygosity and the clinical phenotype, we found it necessary to exclude some of these studies. Two studies were removed because they did not rule out secondary causes of iron overload that were likely to be common in their subjects (Bartolo et al, 1998; Press et al, 1998). A third study was removed because the population was restricted to iron-overloaded individuals without manifestations and also included first-degree relatives of probands (Sham et al., 2000). Two additional studies were removed because they might have included cases reported in an earlier data set (Bacon et al., 1999; Barton et al., 2000) and/or because the inclusion of some HLA-identical siblings of probands could affect genotype frequencies (Bacon et al., 1999). Once these studies were removed, heterogeneity was greatly reduced. Table 3-2 shows the remaining five studies, one of which (Beutler et al., 1996) probably had defined cases adequately, but the manuscript was not sufficiently clear to be sure. The four remaining study estimates were homogeneous, and the summary estimate of the clinical sensitivity was 87 percent (95 percent CI 80 to 94%). Raw data from all 10 studies are available in Appendix A. Exact confidence intervals for individual studies were computed using the binomial distribution (True Epistat, Texas).

Table 3-2 Studies That Can be Used to Compute Clinical Sensitivity of HFE Testing for the Iron Overload Phenotype among Non-Hispanic Caucasians in the U.S.

Among the five studies shown in Table 3-2, none stratify their results according to whether or not the primary iron overload phenotype was present. Instead, cases were defined according to more limited criteria; namely, measurement of iron indices that confirmed iron overload biochemically. Column 3 of Table 3-2 lists the clinical sensitivity as reported in each of the five studies. It was possible to perform further calculations in four of these studies using supplementary data from the study to more rigorously define the clinical phenotype. Column 5 shows the revised clinical sensitivity for the four remaining studies. In the three where more rigorous definitions were used in this analysis (Studies 3, 6 and 5), the revised clinical sensitivity was always higher.

Earlier in this section, an “ideal study” was described that would provide data to define clinical sensitivity. That study would follow a population-based genotyped cohort through life to determine the proportion of C282Y homozygotes that develops clinical manifestations. Such a study is not possible. Instead, the available studies (Table 3-2) use a combination of biochemical and tissue analyses to characterize the extent of iron overload as a surrogate for the clinical phenotype. In addition, many of the individual studies did report some clinical manifestations, but no study separately provided the proportion of these that was homozygous. The current analysis demonstrates that the more rigorously the extent of iron overload is defined, the closer its relationship to C282Y homozygosity becomes. Clinical sensitivity is known to be less than 100 percent, because a small proportion of individuals with the clinical phenotype is known not to be homozygous for C282Y mutation. Thus, clinical sensitivity of C282Y homozygosity for the clinical phenotype is likely to be at least as high as 87 percent but also must be several percentage points less than 100 percent.

Limitations and strengths of this analysis
The reliability of estimating clinical sensitivity of C282Y homozygosity for the primary iron overload phenotype is limited, because the number of acceptable studies (four) and the number of patients studied (318) is small. In addition, all four studies include some individuals who did not have clinical manifestations (e.g., liver damage), a key component of the clinical phenotype. No study included only clinically affected individuals, and none provided a separate estimate for the clinically affected subset. The strength of this analysis is in showing that when the studies are restricted to a more rigorous definition of iron overload, the clinical sensitivity increases. The clinical sensitivity of C282Y homozygosity may even higher than that found in the present analysis.

Gap in knowledge The clinical sensitivity of C282Y homozygosity in individuals with clinical manifestations and documented iron overload has not yet been defined. Currently, our estimate of clinical sensitivity of C282Y homozygosity testing is based mainly on individuals with documented biochemical iron overload who may, or may not, have clinical manifestations. While this is likely to a reasonable approximation, it would be worthwhile to attempt to obtain a more appropriate group for analysis to confirm our estimate.

Figure 3-1 Estimated Clinical Sensitivity of C282Y Homozygosity in Non-Hispanic Caucasians in the U.S. The 10 studies identified in our literature search and summarized in Appendix A, are ordered from lowest to highest clinical sensitivity. The study number (from Appendix A, Table 3-3) is located on the horizontal axis, and the clinical sensitivity (open circle) and associated 95 percent confidence intervals (thin vertical lines) are shown on the vertical-axis. Only four of these studies are used in computing the revised estimates (bolded circles and thick vertical lines). In three instances, the estimates are revised (studies 3,4 and 9), and both the original (thin) and revised (thick) estimates are provided. The horizontal dashed lines indicate the overall revised consensus estimate of the clinical sensitivity (bold horizontal line) and 95 percent confidence intervals (thin horizontal lines).

Clinical Specificity in Other Racial/Ethnic Groups
Hispanic Caucasians There is limited genotype information for Hispanic Caucasians with a clinical diagnosis of HHC. One study from Mexico (Ruiz-Arguell et al., 2000) identified six individuals, none of whom were homozygous for C282Y (two were heterozygotes).

Blacks/African Americans There is limited genotype information for Blacks/African Americans with a clinical diagnosis of HHC. One study from Zimbabwe (Gangaidzo et al., 1999) identified 14 suspected cases of HHC by autopsy. None of the 28 chromosomes carried a C282Y mutation.

Asians/Asian Americans There is limited genotype information for Asians/Asian Americans with a clinical diagnosis of HHC. Two studies (Tsui et al, 2000 and Shiono et al., 2001) identified 12 and 11 cases with clinical findings suggestive of HHC. None of the 46 chromosomes studies carried a C282Y mutation.

Table 3-3. Studies Reporting Frequencies of the C282Y Homozygous Genotype in Non-Hispanic Caucasians in the United States with Primary Iron Overload.

^a Definitions of cases
Study 1 = liver biopsy with hepatic stainable iron of 2+
Study 2 = liver biopsy with elevated hepatic stainable iron
Study 3 = Elevated TS at least twice in the absence of other known causes of IO.
Study 4 = Ranges from elevated TS and serum ferritin to confirmation by liver biopsy or therapeutic phlebotomy
Study 5 = Elevated TS at least twice in the absence of other known causes of IO.
Study 6 = Ranges from elevated TS and serum ferritin to confirmation by liver biopsy or therapeutic phlebotomy
Study 7 = Ranges from elevated TS and serum ferritin to confirmation by liver biopsy or therapeutic phlebotomy.
Study 8 = At least 2 of 4 IO criteria (HIC >4,500 ug/g, HII >2.0, 3-4+ stainable iron, >4g mobilizable iron).
Study 9 = Liver biopsy or quantitative phlebotomy (HII >1.9 or removal of >5g mobilizable iron).
Study 10 = Liver biopsy with 3-4+ hepatic stainable iron or HII >1.9 or HLA identity to a proband.

Reference numbers used for Tables 3-2 and 3-3 and Figure 3-1.
1.       Press et al., 1998
2.       Bartolo et al., 1998
3.       Barton et al., 1997
4.       Sham et al., 2000
5.       Barton et al., 2000
6.       Sham et al., 1997
7.       Beutler et al., 1996
8.       Feder et al., 1996
9.       Brandhagen et al., 2000
10.     Bacon et al., 1999

Bacon BR, Olynyk JK, Brunt EM, Britton RS, Wolff RK. 1999. HFE genotype in patients with hemochromatosis and other liver diseases. Ann Intern Med 130:953-62.

Barton JC, Shih WW, Sawada-Hirai R, Acton RT, Harmon L, Rivers C, Rothenberg BE. 1997. Genetic and clinical description of hemochromatosis probands and heterozygotes: evidence that multiple genes linked to the major histocompatibility complex are responsible for hemochromatosis. Blood Cells Mol Dis. 23:135-145.

Barton JC, Bertoli LF, Rothenberg BE. 2000. Peripheral blood erythrocyte parameters in hemochromatosis: evidence for increased erythrocyte hemoglobin content. J Lab Clin Med 135:96-104.

Bartolo C, McAndrew PE, Sosolik RC, Cawley KA, Balcerzak SP, Brandt JT, Prior TW. 1998. Differential diagnosis of hereditary hemochromatosis from other liver disorders by genetic analysis: gene mutation analysis of patients previously diagnosed with hemochromatosis by liver biopsy. Arch Pathol Lab Med. 122:633-637.

Berlin JA, Laird NM, Sacks HS, Chalmers TC. 1989. A comparison of statistical methods for combining event rates from clinical trials. Stat Med 8:141-151.

Beutler E, Gelbart T, West C, Lee P, Adams M, Blackstone R, Pockros P, Kosty M, Venditti CP, Phatak PD, Seese NK, Chorney KA, Ten Elshof AE, Gerhard GS, Chorney M. 1996. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis. 22:187-194.

Brandhagen DJ, Fairbanks VF, Baldus WP, Smith CI, Kruckeberg KE, Schaid DJ, Thibodeau SN. 2000. Prevalence and clinical significance of HFE gene mutations in patients with iron overload. Am J Gastroenterol 95:2910-2914.

Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Jr., Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Wolff RK, et al. 1996. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399-408.

Gangaidzo IT, Moyo VM, Saungweme T, Khumalo H, Charakupa RM, Gomo ZA, Loyevsky M, Stearman R, La Vaute T, Enquist EG, Rouault TA, Gordeuk VR. 1999. Iron overload in urban Africans in the 1990s. Gut 45:278-283.

Press RD, Flora K, Gross C, Rabkin JM, Corless CL. 1998. Hepatic iron overload: direct HFE (HLA-H) mutation analysis vs quantitative iron assays for the diagnosis of hereditary hemochromatosis. Am J Clin Pathol 109:577-584.

Ruiz-Arguelles GJ, Garces-Eisele J, Gelbart T, Monroy-Barreto M, Reyes-Nunez V, Juarez-Morales JL, de Lourdes Gonzalez-Garrido M, Ramirez-Cisneros FJ, Gallegos-Antunez D. 2000. Analysis of HFE-codon 63/282 (H63D/C282Y) gene variants in mexican mestizos. Blood donors and patients with hereditary hemochromatosis. Arch Med Res 31:422-424.

Sham RL, Ou CY, Cappuccio J, Braggins C, Dunnigan K, Phatak PD. 1997. Correlation between genotype and phenotype in hereditary hemochromatosis: analysis of 61 cases. Blood Cells Mol Dis 23:314-320.

Sham RL, Raubertas RF, Braggins C, Cappuccio J, Gallagher M, Phatak PD. 2000. Asymptomatic hemochromatosis subjects: genotypic and phenotypic profiles. Blood 96:3707-3711.

Shiono Y, Ikeda R, Hayashi H, Wakusawa S, Sanae F, Takikawa T, Imaizumi Y, Yano M, Yoshioka K, Kawanaka M, Yamada G. 2001. C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan. Intern Med 40:852-856.

Tsui WM, Lam PW, Lee KC, Ma KF, Chan YK, Wong MW, Yip SP, Wong CS, Chow AS, Lo ST. 2000. The C282Y mutation of the HFE gene is not found in Chinese haemochromatotic patients: multicentre retrospective study. Hong Kong Med J 6:153-158.